Bio-Rad Laboratories, Inc. Omnition Single-Cell Analysis Software

This analysis pipeline is designed to analyze single-cell ATAC-Seq data and combinatorial single-cell ATAC-Seq data. The pipeline is built using a combination of Nextflow, Conda environments, and Docker containers. To accommodate user environments, the pipeline containers are also compatible with Singularity.

Dependencies

• This software has been tested on the following Linux operating systems: 64-bit CentOS 7 and 8, and Ubuntu 18.04.6, 20.04 LTS, 21.04, and 21.10.
  • The software may run on additional Linux distrubitions and/or versions beyond these if they are able to run the dependencies and versions listed below, but these are not officially supported.
• Internet connection
• Nextflow (21.04.0-22.10.3)
• Docker (>=20.10.7) or Singularity (>=3.6.4)

NOTE: If using Docker, your USER must be added to the docker root user group before executing the pipeline. On shared systems, such as HPC clusters, this may not be possible due to security risks and the pipeline should be executed using the Singularity profile (default) instead.

NOTE: The user must verify with their system administrator that Docker or Singularity is available before using Omnition.

Quick Start

Configure Parameters

The pipeline requires a YAML- or JSON-formatted file listing assay-specific parameters when not running the test data. A detailed description of the file contents, including the structure, are provided below. Assay parameters must be nested under the appropriate assay name (atac) using indentation and only one value may be listed per line. File paths must be relative to the directory where the analysis is being executed. If specifying multiple files, you must create a new nested level of parameters. See below for an example of how to nest parameters as part of a mixed species analysis.

Parameters

NOTE: When configuring bead and cell calling settings, priority will be as follows:

1. Sample-specific settings within an assay-specific section
2. Assay-specific settings
3. Global settings
4. Default settings

Global

• outputDir (default: "./results"): Location to deposit final results and reports.

ATAC

• workflow: Type of workflow to be executed. Must be "reference", "analysis", or "full".

  • "reference": Only perform the reference-generation portion of the given assay
    • Required parameters:
      • reference
        • directory
        • fasta
        • gtf
    • Optional parameters:
      • blocklist
      • mixed
      • barcodedTn5
      • tssWindowSize
  • "analysis": Only perform the analysis portion of the given assay
    • Required parameters:
      • input
      • reference
      • blocklist
    • Optional parameters:
      • mitoContig
      • mixed
      • barcodedTn5
      • ti
      • barcodedTn5Config
      • i7asti
      • tiread
      • tssWindowSize
      • mergeMethod
      • qualityThreshold
      • barcode
        • force
      • trim
      • sortSize
      • rounding
      • maxInsertSize
      • tierroroverride
      • overrides
    • NOTE: This workflow assumes that references have already been generated and will error if they are not found.
  • "full": Perform both reference-generation and analysis portions of the given assay
    • Required parameters:
      • reference
        • directory
        • fasta
        • gtf
      • input
      • blocklist
    • Optional parameters:
      • mitoContig
      • mixed
      • barcodedTn5
      • ti
      • barcodedTn5Config
      • i7asti
      • tiread
      • tssWindowSize
      • mergeMethod
      • qualityThreshold
      • barcode
        • force
      • trim
      • sortSize
      • maxInsertSize
      • tierroroverride
      • overrides

• reference:

  • directory: Directory path to write newly-generated reference files to and/or containing pre-generated references.
    • Required.
    • Must be quoted.
  • fasta: File path(s) to reference FASTA file(s).
    • Required.
    • Optionally gzip compressed; must be quoted.
  • gtf: File path(s) to reference GTF file(s).
    • Required.
    • Optionally gzip compressed; must be quoted.
  • blocklist: File path(s) to reference blocklist BED file(s).
    • Optional. Must be quoted.

• input: Directory path containing raw FASTQ files.

  • Required.
  • Must be gzip compressed; must be quoted.

• mitoContig: The name of the mitochondrial contig in the reference.

  • Optional.
  • DEFAULT: "MT".

• mixed: Boolean value indicating if the mixed species workflow should be used.

  • Optional.
  • If false: Only allows a single FASTA and GTF file to be provided for reference workflow.
  • If true: Requires two FASTA and GTF files to be provided for reference workflow.
  • DEFAULT: false.

• barcodedTn5: Boolean value indicating if the samples have tagmentation indexes.

  • Optional.
  • If false: Samples are assumed to not have tagmentation indexes.
  • If true: All samples are assumed to have tagmentation indexes and read 2 is parsed for the index. Index reports are generated.
  • DEFAULT: false.

• ti: A list of user defined TI sequences.

  • Optional.
  • See ATAC Combinatorial Config file below for example of formatting.
  • DEFAULT: Presets are present in conf/atac_preset.config.

• barcodedTn5Config: File path to a CSV file that specifies the TIs and FASTQs assigned to each sample.

  • Optional.
  • DEFAULT: None.
  • If barcodedTn5 is set to true, and no config file is provided, the default behavior is to merge all TIs and FASTQs as a single sample. Only include TIs present in a given experiment as unused TIs that are included in the config can lead to errors. Must be formatted with these three columns:
  • Sample The name of the sample.
  • Fastq The name given to the fastq file pair.
  • TI: The name of the TI used (see conf/atac_preset.config for list of preset TIs).

• i7asti: Boolean value indicating whether the I7 index should be treated as a tagmentation index.

  • Optional.
  • Requires barcodedTn5 to be true.
  • DEFAULT: false.

• tiread: The read that contains the TI sequence, assuming the TI is not in the i7 sequence.

  • Optional.
  • Requires barcodedTn5 to be true and i7asti to be false.
  • One of "r1" or "r2".
  • DEFAULT: "r1".

• tssWindowSize: The full window size in bases around TSS for TSS enrichment score calculations.

  • Optional.
  • Must be an even integer greater than 0.
  • DEFAULT: 4000.

• mergeMethod: The read(s) in a pair to use for determining the transposase insertion site used in bead merging.

  • Optional.
  • One of "r1", "r2", or "both".
  • DEFAULT: "both".

• qualityThreshold: Minimum MAPQ for a read to be included in the analysis.

  • Optional.
  • DEFAULT: 30.

• barcode: Settings related to knee calling configuration.

  • Optional.
  • force: The number of barcodes to return. This will bypass all knee calling algorithms and return the top n number of barcodes based on unique reads.
    • An integer greater than zero.
    • DEFAULT: None. Omnition will determine the number of cells in each sample.

• trim: Number of bases to trim from the 5' end of R2 read.

  • Optional.
  • An integer greater than or equal to 0.
  • DEFAULT: 0.

• sortSize: Set the sort collection size ratio for MarkDuplicates.

  • Optional.
  • A floating point number greater than 0.
  • This parameter is to be used when the module runs out of memory due to very high duplication rate.
  • Lower the number to make memory footprint size more manageable. Recommended trial value is 0.01 in case of memory errors.
  • DEFAULT: 0.25.

• rounding: Rounds insert sites to the nearest 10, 100, or 1000 bases before performing bead merging.

  • Optional.
  • One of 10, 100, or 1000.
  • DEFAULT: 0; no rounding is performed.

• maxInsertSize: The largest insert that Omnition will recognize.

  • Optional.
  • An integer greater than 100.
  • DEFAULT: 2000.

• tierroroverride: Boolean value.

  • If set to true, the pipeline will ignore errors in the TI configuration file. Only fastq-TI combinations specified in the config file will be used. All others observed in the sequencing data will be ignored.
  • DEFAULT: false.

• overrides: (non-combinatorial) Sample-specific settings for overriding barcode calling and bead merging configuration.

  • <sample_id>: The sample_id of the sample to be overriden.
    • barcode: See above for description.
      • force
    • trim: See above for description.
    • mergeMethod: See above for description.

• overrides: (combinatorial) FASTQ+TI-specific settings for overriding barcode calling and deconvoulution configuration. Overrides can be set on the FASTQ level or on the FASTQ+TI level. Overrides set on the FASTQ level will be applied to all FASTQ+TI combinations coming from that FASTQ file.

  • <sample_id>: The FASTQ name of the fastq to be overriden.
    • barcode: See above for description.
      • force
    • trim: See above for description.
    • mergeMethod: See above for description. Example: If the FASTQ files were named SampleA_S1_R1_001.fastq.gz and SampleA_S1_R1_001.fastq.gz, the FASTQ name would be SampleA_S1.
  • <sample_id>: The FASTQ name of the fastq to be overriden.
    • <TI_name>: The TI name of the TI to be overriden. Example: TI12.
      • barcode: See above for description.
        • force
      • trim: See above for description.
      • mergeMethod: See above for description.

Examples

parameters_atac.yaml (ATAC-Seq)

atac:
  workflow: "full"
  input: "test/data/atac/normal/"
  reference: 
    directory: "test/references/atac/"
    fasta:
        species1: "test/references/atac/final_hg38.fa.gz"
        species2: "test/references/atac/final_mm10.fa.gz"
    gtf:
        species1: "test/references/atac/final_hg38.gtf.gz"
        species2: "test/references/atac/final_mm10.gtf.gz"
  mixed: true 

parameters_catac.yaml (Combinatorial ATAC-Seq Superloading)

atac:
  workflow: "full"
  input: "test/data/atac/combinatorial/"
  reference: 
    directory:"test/references/atac/"
    fasta:
        species1: "test/references/atac/final_hg38.fa.gz"
        species2: "test/references/atac/final_mm10.fa.gz"
    gtf:
        species1: "test/references/atac/final_hg38.gtf.gz"
        species2: "test/references/atac/final_mm10.gtf.gz"
  mixed: true
  barcodedTn5: true
  ti:
    ti1: "AAAGAA"
    ti2: "TTTGGG"
  barcodedTn5Config: "test/config/atac/atac_mixed_TIs.csv"
  tiread: "r1"

NOTE: Removing the barcodedTn5Config parameter from above will change the configuration to Combinatorial ATAC-Seq Superloading.

atac_mixed_TIs.csv

Sample,Fastq,TI
SampleA,Cycler_S3_newindex,mycustomti1
SampleB,Cycler_S3_newindex,mycustomt12

Custom Transposition Index (TI) definition for combinatorial ATAC-Seq

To use custom TIs, add the TI sequences to your yaml config file. See parameters_catac.yaml, above, for an example.

Custom TIs must be of equal length and must be located in one of the following locations:

• The 5' end of R2 (see parameter tiread: r2)
• Immediately after the bead barcode in R1 (set parameter tiread: r1)
• In the i7 sequencing barcode (see parameter i7asti: true).

For TIs located in the 5' end of R2, use the trim parameter to remove all bases upstream of the TI sequence.

References

Omnition is compatible with Human/GRCh38 and Mouse/GRCm39 references from ENSEMBL. It is not compatible with other species or references produced by NCBI.

Execution

The pipeline may be executed using either Singularity (default) or Docker. Upon execution, Nextflow will download the pipeline from GitHub. The code may be found in ~/.nextflow/assets/BioRadOpenSource/omnition/ after download. Omnition will download the Singularity images or Docker containers as needed.

Please note the use of single - and double -- in the execution commands. Arguments with a single - in front are Nextflow arguments and those with double -- are user-defined parameters.

NOTE: Nextflow gives each pipeline run a randomly generated name formatted as [adjective_name]. Names are taken from those listed in the following code (https://github.com/nextflow-io/nextflow/blob/master/modules/nextflow/src/main/groovy/nextflow/util/NameGenerator.groovy). These run names are generated by Nextflow and not endorsed by Bio-Rad.

After completion, reports can be found in the report/ subdirectory and intermediate files can be found in the Sample_Files/ subdirectory. Additionally, a Nextflow cache directory (.nextflow/) and working directory (work/) will be created in the directory where Nextflow was executed.

Nextflow Functionality

Resuming an Analysis

Interrupted or failed analyses can be restarted from their stopping point by using the -resume flag in the pipeline execution commands. Deleting the cache (.nextflow/) or working directory (work/) will clear the cache and disable the -resume functionality.

Diagnostics

Pipeline diagnostic reports generated by Nextflow will be produced by default by Omnition. For each analysis, a pipeline_info directory will be generated in the --outputDir and populated with a Nextflow execution report, trace report, and timeline report. For more information on these reports, see the Nextflow documentation.

Singularity

nextflow run BioRadOpenSource/omnition -params-file <path to parameters.yaml> --outputDir <output directory path>

Docker

nextflow run BioRadOpenSource/omnition -params-file <path to parameters.yaml> --outputDir <output directory path> -profile docker

Test Data

There are two test profiles available to run with the pipeline. The profile demo_atac runs with the settings specified in parameters_atac.yaml, above. The profile demo_catac runs with the settings specified in the parameters_catac.yaml example, above. The parameters used in these different testing profiles can be found in conf/test.config.

Below are examples of how to run the different testing profiles while executing the pipeline with Docker (-profile docker) or Singularity (-profile standard). The test type is then specified separated by a comma in the -profile flag.

Docker:

nextflow run BioRadOpenSource/omnition --outputDir <output directory path> -profile docker,demo_atac
nextflow run BioRadOpenSource/omnition --outputDir <output directory path> -profile docker,demo_catac

Singularity:

nextflow run BioRadOpenSource/omnition --outputDir <output directory path> -profile standard,demo_atac
nextflow run BioRadOpenSource/omnition --outputDir <output directory path> -profile standard,demo_catac

Appendix

Resource Allocation

Omnition is composed of a series of “processes” that connect to form a pipeline. Each process is allocated a portion of the host system’s CPU and RAM when Nextflow’s scheduler launches a processing task. Nextflow’s scheduler launches processes in parallel when the system’s capacity permits it.

Bio-Rad has provided default resource allocations for the processes in Omnition to optimize performance on a system with 16 CPU and 64 GB RAM. If a process fails due to insufficient resources, it will be retried a maximum of two times with each retry requesting double the resources of the previous one. Should the process fail after a second retry, no further retries will be carried out. Each process is assigned two labels that dictate the CPU and memory allocation to that process. The default labels are defined in the tables below.

CPU Allocation Labels

Label	CPUs
cpu_xsmall	1
cpu_small	2
cpu_medium	4
cpu_large	8
cpu_xlarge	16

Memory Allocation Labels

Label	RAM (GB)
memory_xxsmall	0.9375
memory_xsmall	3.75
memory_small	7.5
memory_medium	15
memory_large	30
memory_xlarge	60

Default Process Labels

Process	CPUs	RAM
AGGREGATE_METRICS	cpu_small	memory_xsmall
ANNOTATE_FRAGMENTS	cpu_medium	memory_medium
ARCHR	cpu_small	memory_xlarge
ARCHR_REFERENCE	cpu_small	memory_large
ASSEMBLE_BASIC_QC	cpu_medium	memory_xxsmall
ASSEMBLE_FRAGMENTS	cpu_xsmall	memory_xsmall
BEAD_FILT_SUMMARY	cpu_small	memory_xxsmall
BUILD_REPORT_CONTENTS	cpu_medium	memory_xxsmall
BWA_ALIGNMENT	cpu_xlarge	memory_xlarge
BWA_INDEX	cpu_small	memory_large
CALCULATE_BEADS_PER_DROP	cpu_xsmall	memory_xxsmall
CALCULATE_INSERT_SIZE_METRICS	cpu_small	memory_xsmall
CALL_PEAKS	cpu_xsmall	memory_medium
CHECK_MITO_CONTIG	cpu_xsmall	memory_xsmall
CHECK_TI_COUNTS	cpu_xsmall	memory_xxsmall
CHECK_TI_COUNTS_SUPERLOADED	cpu_xsmall	memory_xxsmall
CLEAN_PEAKS	cpu_xsmall	memory_xsmall
COMBINE_BLOCKLISTS	cpu_xsmall	memory_xxsmall
COMBINE_REFERENCES	cpu_small	memory_xxsmall
COMPILE_ALIGNMENTS	cpu_large	memory_xsmall
COMPILE_FRAGMENTS	cpu_large	memory_medium
COMPILE_QC_STATS	cpu_large	memory_xxsmall
COMPUTE_DECONVOLUTION_STAT_CHR	cpu_medium	memory_medium
COMPUTE_TSS_MATRIX	cpu_small	memory_xxsmall
CUTADAPT_HEADCROP	cpu_medium	memory_medium
DEAD	cpu_small	memory_xxsmall
DETERMINE_BARCODE_ALLOWLIST	cpu_medium	memory_small
DETERMINE_BARCODE_MERGES	cpu_medium	memory_small
FASTQC	cpu_small	memory_small
FILTER_BLOCKLISTS	cpu_xsmall	memory_xxsmall
FILTER_REFERENCES	cpu_xsmall	memory_xxsmall
FINAL_BAM_MERGE	cpu_xlarge	memory_xxsmall
FINAL_FRAG_MERGE	cpu_medium	memory_xsmall
FINAL_QC_SE	cpu_medium	memory_xlarge
FORMAT_BLOCKLIST	cpu_xsmall	memory_xxsmall
FRACTION_OF_READS_IN_PEAKS	cpu_xsmall	memory_xsmall
FRACTION_OF_READS_IN_TSS	cpu_xsmall	memory_xsmall
GENERATE_EMPTY_BLOCKLIST	cpu_xsmall	memory_xxsmall
GENERATE_GENOME_SIZES	cpu_medium	memory_xxsmall
GENERATE_REPORT	cpu_small	memory_small
GENERATE_TSS_WINDOWS	cpu_medium	memory_xxsmall
GUNZIP	cpu_xsmall	memory_xxsmall
MAKE_COUNT_MATRIX	cpu_xsmall	memory_xlarge
MARK_DUPLICATES	cpu_medium	memory_xlarge
MERGE_LANES	cpu_xsmall	memory_xxsmall
MERGE_REANN_READ_COUNTS	cpu_small	memory_xxsmall
PUBLISH_PARAMETERS	cpu_medium	memory_xxsmall
REANNOTATE_BAM	cpu_medium	memory_xxsmall
REANNOTATE_FRAGMENTS	cpu_medium	memory_medium
SEQUENCE_SATURATION	cpu_medium	memory_medium
SPLIT_BAM	cpu_xlarge	memory_xsmall
SPLIT_FASTQ	cpu_xsmall	memory_xxsmall
SUMMARIZE_ALIGNMENTS	cpu_medium	memory_xsmall
SUMMARIZE_MIXED_SPECIES	cpu_xsmall	memory_xxsmall
TAG_BARCODES	cpu_xlarge	memory_xlarge
TI_DEAD_CONFIG	cpu_xsmall	memory_xxsmall
TI_ERROR_CHECK	cpu_xsmall	memory_xxsmall
TI_WARNING_MESSAGES	cpu_xsmall	memory_xsmall
TSS_ENRICHMENT	cpu_xsmall	memory_xxsmall
VALIDATE_TI_CONFIG	cpu_xsmall	memory_xxsmall

Adjusting Resource Allocation

To change the resource allocation for a given resource tier (e.g., increase memory for the memory_xlarge label), create a configuration file like the one below and save it as resources.config:

process{ 
    withLabel: memory_xlarge {  
        memory = 128.GB  
        time = 24.h  
    }
} 

This file should then be saved and passed to Omnition when running the software, for example:

nextflow run BioRadOpenSource/omnition -params-file analysis.yaml -profile standard –c resources.config

The resource allocations in resources.config will then override the default settings.

ATAC BAM File Tags

This pipeline places a number of tags on the bam files produced in the workflow. Below is a table defining them.

Tag	Type	First Annotated	Description
AS	i	process BWA_ALIGNMENT	Assigned by BWA and conforms to SAM specifications: Alignment score from the aligner. Lay definition: See previous sentence.
NM	i	process BWA_ALIGNMENT	Assigned by BWA. The edit distance in the alignment.
MC	Z	process BWA_ALIGNMENT	Compressed representation of the alignment's mate in the [CIGAR format].(https://www.drive5.com/usearch/manual/cigar.html)
MD	Z	process BWA_ALIGNMENT	Mismatching positions and bases.
XS	i	process BWA_ALIGNMENT	Alignment scores for suboptimal alignments.
XA	Z	process BWA_ALIGNMENT	Suboptimal alignment hits. Format: (chr,pos,CIGAR,NM).
XB	Z	process BWA_ALIGNMENT	The bead barcode sequence for a read.
PG	Z	process MARK_DUPLICATES	Indicates MarkDuplicates was executed.
DI	i	process MARK_DUPLICATES	The duplicate set index as assigned by MarkDuplicates.
DB	Z	process REANNOTATE_BAM	The cell barcode.

Blocklist

NOTE: Bio-Rad has adopted the term "blocklist” as a replacement for “blacklist.”

The ATAC analysis workflow will ignore alignments to blocklisted regions within the genome during bead merging. Bio-Rad does not provide blocklists. File name must be in the format SPECIESNAME.blocklist.bed where SPECIESNAME is the same species name used for the fasta and gtf files. Blocklists must be formatted as a three column BED file with feature names matching those in the reference genome FASTA and reference GTF:

chr10      0       45700
chr10      38481300        38596500
chr10      38782600        38967900
chr10      39901300        41712900
chr10      41838900        42107300
chr10      42279400        42322500
chr10      126946300       126953400
chr10      133625800       133797400

A blocklist is not required for the pipeline to execute. If one is not provided, an empty one is generated at runtime.

When doing a mixed species run, up to two blocklists may be supplied.

Publicly available blocklists are available here. The feature names need to be updated to match those of the reference being used.

Benchmarks

The dataset used for quantification was a mixed species run with 1,262,814,294 total paired reads which were downsampled to 4 different levels (100%, 75%, 50%, 25%, 10%) when run through the analysis workflow. Without downsampling the pipeline called 32,812 total cells.

A system with 16 CPUs (Intel Xeon Cascade Lake) and 128 GB RAM was (AWS r5d.4xlarge) used for the runs used to generate these results.

Reference workflow

name	realtime (run time)	peak_rss (peak RAM usage)
ARCHR_REFERENCE	9m 2s	11.3 GB
BWA_INDEX	2h 25s	8.2 GB
COMBINE_REFERENCES	24s	41.5 MB
FILTER_BLOCKLISTS	18.5s	66.7 MB
FILTER_REFERENCES	26.3s	63.8 MB
FORMAT_BLOCKLIST	4s	57.3 MB
GENERATE_GENOME_SIZES	23.1s	31.2 MB
GENERATE_TSS_WINDOWS	9.8s	137.6 MB
GUNZIP_FASTA	27.2s	58.4 MB
GUNZIP_GTF	9.3s	59.1 MB

Analysis workflow

% of sample reads used	total paired reads
100	1,262,814,294
75	947,110,720
50	631,407,146
25	315,703,572
10	126,281,428

name	realtime (run time)	peak_rss (peak RAM usage)	% of initial sample reads used
AGGREGATE_METRICS	3m 12s	767.2 MB	100
AGGREGATE_METRICS	2m 47s	759.1 MB	75
AGGREGATE_METRICS	2m 6s	706.3 MB	50
AGGREGATE_METRICS	1m 39s	643.3 MB	25
AGGREGATE_METRICS	2m 29s	932.1 MB	10
ANNOTATE_FRAGMENTS	9s	957.4 MB	100
ANNOTATE_FRAGMENTS	9s	996.6 MB	75
ANNOTATE_FRAGMENTS	9s	982.8 MB	50
ANNOTATE_FRAGMENTS	9s	650.1 MB	25
ANNOTATE_FRAGMENTS	7s	353.3 MB	10
ARCHR	1h 51m 47s	68.4 GB	100
ARCHR	1h 50m 11s	61.2 GB	75
ARCHR	1h 42m 24s	76.3 GB	50
ARCHR	1h 31m 43s	70.1 GB	25
ARCHR	1h 25m 14s	51.1 GB	10
ASSEMBLE_BASIC_QC	4s	352 MB	100
ASSEMBLE_BASIC_QC	4s	359.7 MB	75
ASSEMBLE_BASIC_QC	4s	351.9 MB	50
ASSEMBLE_BASIC_QC	4s	360.4 MB	25
ASSEMBLE_BASIC_QC	4s	361.9 MB	10
ASSEMBLE_FRAGMENTS	9s	998 MB	100
ASSEMBLE_FRAGMENTS	9s	989.6 MB	75
ASSEMBLE_FRAGMENTS	9s	99.1 MB	50
ASSEMBLE_FRAGMENTS	9s	99.8 MB	25
ASSEMBLE_FRAGMENTS	8s	98.9 MB	10
BEAD_FILT_SUMMARY	11.4s	14.8 MB	100
BEAD_FILT_SUMMARY	9.1s	14.6 MB	75
BEAD_FILT_SUMMARY	6.3s	14.5 MB	50
BEAD_FILT_SUMMARY	3.2s	14.6 MB	25
BEAD_FILT_SUMMARY	2.4s	14.7 MB	10
BUILD_REPORT_CONTENTS	2.5s	122.4 MB	100
BUILD_REPORT_CONTENTS	2.6s	121.9 MB	75
BUILD_REPORT_CONTENTS	2.5s	123.2 MB	50
BUILD_REPORT_CONTENTS	2.5s	122.4 MB	25
BUILD_REPORT_CONTENTS	2.6s	121.8 MB	10
BWA_ALIGNMENT	38m 22s	29.6 GB	100
BWA_ALIGNMENT	43m 50s	29.6 GB	75
BWA_ALIGNMENT	9m 54s	29.2 GB	50
BWA_ALIGNMENT	9m 44s	23 GB	25
BWA_ALIGNMENT	33m 19s	16.5 GB	10
CALCULATE_BEADS_PER_DROP	2s	37.3 MB	100
CALCULATE_BEADS_PER_DROP	2s	36.3 MB	75
CALCULATE_BEADS_PER_DROP	2s	36.4 MB	50
CALCULATE_BEADS_PER_DROP	2s	35.5 MB	25
CALCULATE_BEADS_PER_DROP	3s	74.8 MB	10
CALCULATE_INSERT_SIZE_METRICS	25m 58s	1.1 GB	100
CALCULATE_INSERT_SIZE_METRICS	20m 8s	1.1 GB	75
CALCULATE_INSERT_SIZE_METRICS	13m 54s	972.2 MB	50
CALCULATE_INSERT_SIZE_METRICS	8m 29s	667.4 MB	25
CALCULATE_INSERT_SIZE_METRICS	4m 5s	1.4 GB	10
CALL_PEAKS	44m 6s	3.3 GB	100
CALL_PEAKS	37m 25s	2.7 GB	75
CALL_PEAKS	29m 26s	2.2 GB	50
CALL_PEAKS	17m 51s	1.4 GB	25
CALL_PEAKS	9m 9s	736.3 MB	10
CHECK_TI_COUNTS_SUPERLOADED	41m 20s	14.8 MB	100
CHECK_TI_COUNTS_SUPERLOADED	32m 12s	28 MB	75
CHECK_TI_COUNTS_SUPERLOADED	21m 27s	33.4 MB	50
CHECK_TI_COUNTS_SUPERLOADED	10m 35s	32.6 MB	25
CHECK_TI_COUNTS_SUPERLOADED	1m 57s	14.9 MB	10
CLEAN_PEAKS	13.1s	372.7 MB	100
CLEAN_PEAKS	12.4s	415.2 MB	75
CLEAN_PEAKS	12s	409.5 MB	50
CLEAN_PEAKS	10.8s	372.9 MB	25
CLEAN_PEAKS	9.5s	355.7 MB	10
COMPILE_ALIGNMENTS	2h 11m 50s	403.7 MB	100
COMPILE_ALIGNMENTS	1h 56m 13s	435.4 MB	75
COMPILE_ALIGNMENTS	1h 26m 7s	419.7 MB	50
COMPILE_ALIGNMENTS	50m 8s	385.8 MB	25
COMPILE_ALIGNMENTS	18m 34s	630.5 MB	10
COMPILE_QC_STATS	1.1s	13.1 MB	100
COMPILE_QC_STATS	1.1s	11.8 MB	75
COMPILE_QC_STATS	1s	12 MB	50
COMPILE_QC_STATS	1.4s	12.4 MB	25
COMPILE_QC_STATS	958ms	12 MB	10
COMPUTE_DECONVOLUTION_STAT_CHR	9s	996.6 MB	100
COMPUTE_DECONVOLUTION_STAT_CHR	9s	99.5 MB	75
COMPUTE_DECONVOLUTION_STAT_CHR	9s	99.9 MB	50
COMPUTE_DECONVOLUTION_STAT_CHR	9s	99.6 MB	25
COMPUTE_DECONVOLUTION_STAT_CHR	9.9s	99.9 MB	10
COMPUTE_TSS_MATRIX	1h 56s	1.7 GB	100
COMPUTE_TSS_MATRIX	51m 54s	1.5 GB	75
COMPUTE_TSS_MATRIX	44m 37s	1.2 GB	50
COMPUTE_TSS_MATRIX	34m 42s	802.5 MB	25
COMPUTE_TSS_MATRIX	26m 26s	653.2 MB	10
COUNT_BEADS_PER_PARTITION	3.8s	98.5 MB	100
COUNT_BEADS_PER_PARTITION	7.6s	98.3 MB	75
COUNT_BEADS_PER_PARTITION	6s	98 MB	50
COUNT_BEADS_PER_PARTITION	7.1s	99.7 MB	25
COUNT_BEADS_PER_PARTITION	5.6s	98.6 MB	10
CUTADAPT_HEADCROP	8m 31s	4.3 GB	100
CUTADAPT_HEADCROP	7m 49s	4.1 GB	75
CUTADAPT_HEADCROP	5m 10s	4 GB	50
CUTADAPT_HEADCROP	2m 5s	3.3 GB	25
CUTADAPT_HEADCROP	36.7s	991.6 MB	10
DEAD	3h 31m 53s	135.7 MB	100
DEAD	2h 35m 53s	135.6 MB	75
DEAD	1h 43m 36s	135.7 MB	50
DEAD	51m 51s	135.6 MB	25
DEAD	12m 42s	135.7 MB	10
DETERMINE_BARCODE_ALLOWLIST	3m 5s	1.2 GB	100
DETERMINE_BARCODE_ALLOWLIST	2m 13s	1.2 GB	75
DETERMINE_BARCODE_ALLOWLIST	60s	1.1 GB	50
DETERMINE_BARCODE_ALLOWLIST	59s	1.1 GB	25
DETERMINE_BARCODE_ALLOWLIST	41.1s	996.7 MB	10
DETERMINE_BARCODE_MERGES	34.3s	658 MB	100
DETERMINE_BARCODE_MERGES	9.9s	570.5 MB	75
DETERMINE_BARCODE_MERGES	9.7s	369.7 MB	50
DETERMINE_BARCODE_MERGES	9.1s	198.4 MB	25
DETERMINE_BARCODE_MERGES	5s	163 MB	10
FASTQC	2h 19m 22s	596.2 MB	100
FASTQC	1h 44m 49s	609.9 MB	75
FASTQC	1h 10m 15s	602.5 MB	50
FASTQC	36m 25s	577.1 MB	25
FASTQC	8m 50s	568.3 MB	10
FINAL_BAM_MERGE	57.1s	44.2 MB	100
FINAL_BAM_MERGE	59.1s	47.8 MB	75
FINAL_BAM_MERGE	58s	46.8 MB	50
FINAL_BAM_MERGE	58.9s	45.1 MB	25
FINAL_BAM_MERGE	9.9s	44.1 MB	10
FINAL_FRAG_MERGE	59s	43.3 MB	100
FINAL_FRAG_MERGE	58.7s	45.1 MB	75
FINAL_FRAG_MERGE	49.9s	8.7 MB	50
FINAL_FRAG_MERGE	24.8s	43 MB	25
FINAL_FRAG_MERGE	8.1s	55.7 MB	10
FINAL_QC_SE	59.5s	4.7 GB	100
FINAL_QC_SE	48.9s	3 GB	75
FINAL_QC_SE	39.6s	2.6 GB	50
FINAL_QC_SE	36.2s	988.2 MB	25
FINAL_QC_SE	9.8s	762.6 MB	10
FRACTION_OF_READS_IN_PEAKS	2h 2m 32s	522.3 MB	100
FRACTION_OF_READS_IN_PEAKS	1h 39m 30s	553 MB	75
FRACTION_OF_READS_IN_PEAKS	1h 15m 56s	528.1 MB	50
FRACTION_OF_READS_IN_PEAKS	45m 3s	481.4 MB	25
FRACTION_OF_READS_IN_PEAKS	20m 45s	732.7 MB	10
FRACTION_OF_READS_IN_TSS	2h 20m 34s	512.1 MB	100
FRACTION_OF_READS_IN_TSS	1h 54m 39s	548.5 MB	75
FRACTION_OF_READS_IN_TSS	1h 26m 45s	532.8 MB	50
FRACTION_OF_READS_IN_TSS	52m 11s	500.9 MB	25
FRACTION_OF_READS_IN_TSS	24m 9s	775.7 MB	10
GENERATE_REPORTS	11m 55s	8.1 GB	100
GENERATE_REPORTS	9m 32s	7.1 GB	75
GENERATE_REPORTS	6m 22s	5.9 GB	50
GENERATE_REPORTS	2m 48s	1.6 GB	25
GENERATE_REPORTS	2m 11s	4.2 GB	10
MAKE_COUNT_MATRIX	14m 41s	20 GB	100
MAKE_COUNT_MATRIX	12m 56s	18.6 GB	75
MAKE_COUNT_MATRIX	9m 37s	16.4 GB	50
MAKE_COUNT_MATRIX	6m 20s	12.9 GB	25
MAKE_COUNT_MATRIX	3m 31s	13.5 GB	10
MARK_DUPLICATES	28m 31s	48.8 GB	100
MARK_DUPLICATES	9m 1s	42.2 GB	75
MARK_DUPLICATES	9m 49s	37.6 GB	50
MARK_DUPLICATES	7m 4s	35.8 GB	25
MARK_DUPLICATES	1m 9s	31.7 GB	10
MERGE_LANES	7m 30s	65.3 MB	100
MERGE_LANES	8m 22s	53.9 MB	75
MERGE_LANES	2m 28s	40.1 MB	50
MERGE_LANES	2m 56s	41.5 MB	25
MERGE_LANES	3.7s	55.6 MB	10
MERGE_REANN_READ_COUNTS	3.9s	126.8 MB	100
MERGE_REANN_READ_COUNTS	5.9s	127 MB	75
MERGE_REANN_READ_COUNTS	5.5s	126.9 MB	50
MERGE_REANN_READ_COUNTS	6.3s	126.8 MB	25
MERGE_REANN_READ_COUNTS	5.4s	126.9 MB	10
REANNOTATE_BAM	9s	98 MB	100
REANNOTATE_BAM	9s	97.8 MB	75
REANNOTATE_BAM	9s	97.2 MB	50
REANNOTATE_BAM	9s	90.3 MB	25
REANNOTATE_BAM	9s	6.1 MB	10
REANNOTATE_FRAGMENTS	9s	998.2 MB	100
REANNOTATE_FRAGMENTS	9s	985.5 MB	75
REANNOTATE_FRAGMENTS	9s	895.8 MB	50
REANNOTATE_FRAGMENTS	9.5s	583.7 MB	25
REANNOTATE_FRAGMENTS	5s	340.7 MB	10
SEQUENCE_SATURATION	2m 5s	7.5 GB	100
SEQUENCE_SATURATION	1m 52s	6.7 GB	75
SEQUENCE_SATURATION	1m 32s	5.6 GB	50
SEQUENCE_SATURATION	1m 4s	4 GB	25
SEQUENCE_SATURATION	37.6s	2.2 GB	10
SPLIT_BAM	3m 51s	943.4 MB	100
SPLIT_BAM	3m 34s	944 MB	75
SPLIT_BAM	41.5s	933.8 MB	50
SPLIT_BAM	59.9s	924.6 MB	25
SPLIT_BAM	9.7s	798.1 MB	10
SPLIT_FASTQ	2h 9m 36s	67.1 MB	100
SPLIT_FASTQ	1h 42m 17s	64.3 MB	75
SPLIT_FASTQ	59m 57s	65.7 MB	50
SPLIT_FASTQ	28m 28s	65.6 MB	25
SPLIT_FASTQ	6m 12s	63.1 MB	10
SUMMARIZE_ALIGNMENTS	2h 54m 18s	1.3 GB	100
SUMMARIZE_ALIGNMENTS	2h 28m	1.2 GB	75
SUMMARIZE_ALIGNMENTS	1h 43m 13s	940.6 MB	50
SUMMARIZE_ALIGNMENTS	59m 45s	1.4 GB	25
SUMMARIZE_ALIGNMENTS	32m 56s	1.4 GB	10
SUMMARIZE_MIXED_SPECIES	3.1s	96.3 MB	100
SUMMARIZE_MIXED_SPECIES	2.9s	117.3 MB	75
SUMMARIZE_MIXED_SPECIES	3.1s	105.7 MB	50
SUMMARIZE_MIXED_SPECIES	3.1s	100.1 MB	25
SUMMARIZE_MIXED_SPECIES	3.8s	121.9 MB	10
TAG_BARCODES	5m 19s	14.2 GB	100
TAG_BARCODES	7m 48s	14.1 GB	75
TAG_BARCODES	4m 34s	9.7 GB	50
TAG_BARCODES	59.7s	9.5 GB	25
TAG_BARCODES	44.1s	2.1 GB	10
TI_DEAD_CONFIG	3.4s	12.6 MB	100
TI_DEAD_CONFIG	6.7s	59.8 MB	75
TI_DEAD_CONFIG	4.9s	47.9 MB	50
TI_DEAD_CONFIG	5s	54.1 MB	25
TI_DEAD_CONFIG	6.8s	55 MB	10
TSS_ENRICHMENT	4m 23s	16.4 MB	100
TSS_ENRICHMENT	4m 21s	16.4 MB	75
TSS_ENRICHMENT	4m 19s	16.5 MB	50
TSS_ENRICHMENT	4m 17s	16.3 MB	25
TSS_ENRICHMENT	4m 15s	16.3 MB	10